N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-L-phenylalanine ethyl ester as a stable ester-type inhibitor of chymotrypsin-like serine proteases: Structural requirements for potent inhibition of α-chymotrypsin

Article abstract of DOI:10.1021/jm980562h

We introduce a new potent inhibitor, N-[2,2-dimethyl-3-(N-(4- cyanobenzoyl)amino)nonanoyl]n-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure, showed strong inhibitory activities toward bovine α-chymotrypsin, human cathepsin G, and porcine elastase by acting as an acylating agent. The calculated inactivation rate constant (k(inact)) and enzyme-inhibitor dissociation constant (K(i)) against α-chymotrypsin were 0.0028 s^-1 and 0.0045 μM, respectively (k(inact)/K(i) = 630 000 M^-1 s^-1). These kinetic parameters indicate that this inhibitor is one of the most powerful α- chymotrypsin inactivators ever reported. On the basis of structure-activity relationship (SAR) and structure-stability relationship studies of analogues of 3, which were modified in three parts of the molecule, i.e., the 4- cyanophenyl group, β-substituent at the β-amino acid residue, and ester structure, we suggest that the potent inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which enforces specific acyl-enzyme formation, (2) the n-hexyl group at the β-position and 4- cyanophenyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues against different serine proteases such as chymase, cathepsin G, and elastase suggest that these compounds recognize common parts in the active sites among these chymotrypsin-like serine proteases, and 3 is one of the most suitable structures to recognize those common parts. Our results provide an intriguing basis for further developments in the design of a stable ester-based selective serine protease inhibitor.

Full text of DOI:10.1021/jm980562h

312
J . Med. Chem. 1999, 42, 312-323
N-[2,2-Dim eth yl-3-(N-(4-cya n oben zoyl)a m in o)n on a n oyl]-L-p h en yla la n in e Eth yl
Ester a s a Sta ble Ester -Typ e In h ibitor of Ch ym otr yp sin -lik e Ser in e P r otea ses:
Str u ctu r a l Requ ir em en ts for P oten t In h ibition of r-Ch ym otr yp sin
Kiyoko Iijima, J un Katada, Emiko Yasuda, Isao Uno, and Yoshio Hayashi*
Life Science Research Center, Advanced Technology Research Laboratories, Nippon Steel Corporation, 3-35-1 Ida,
Nakahara-ku, Kawasaki 211-0035, J apan
Received October 5, 1998
We introduce a new potent inhibitor, N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanoyl]-
L-phenylalanine ethyl ester (3), which preferentially inhibits serine proteases belonging to a
chymotrypsin superfamily. This inhibitor, despite consisting of a stable ethyl ester structure,
showed strong inhibitory activities toward bovine R-chymotrypsin, human cathepsin G, and
porcine elastase by acting as an acylating agent. The calculated inactivation rate constant
(kinact) and enzyme-inhibitor dissociation constant (Ki) against R-chymotrypsin were 0.0028
s-1 and 0.0045 µM, respectively (kinact/Ki ) 630 000 M-1 s-1). These kinetic parameters indicate
that this inhibitor is one of the most powerful R-chymotrypsin inactivators ever reported. On
the basis of structure-activity relationship (SAR) and structure-stability relationship studies
of analogues of 3, which were modified in three parts of the molecule, i.e., the 4-cyanophenyl
group, â-substituent at the â-amino acid residue, and ester structure, we suggest that the potent
inhibitory activity of 3 is due to the following structural features: (1) the ethyl ester which
enforces specific acyl-enzyme formation, (2) the n-hexyl group at the â-position and 4-cyanophe-
nyl group which stabilize the acyl-enzyme, and (3) the phenylalanine residue which functions
for the specific recognition of S1 site in the enzyme. In particular, the action of 3 as a potent
inhibitor, but poor substrate, can be ascribed largely to the very slow deacylation rate depending
on the structure factors cited in feature 2. The results of inhibition by 3 and its analogues
against different serine proteases such as chymase, cathepsin G, and elastase suggest that
these compounds recognize common parts in the active sites among these chymotrypsin-like
serine proteases, and 3 is one of the most suitable structures to recognize those common parts.
Our results provide an intriguing basis for further developments in the design of a stable ester-
based selective serine protease inhibitor.
In tr od u ction
anobenzoyl)amino)-5-phenylpentanoic anhydride] was a
potent inhibitor of R-chymotrypsin (kinact/Ki ) 242 000
M-1 s-1, kinact ) 0.017 s-1, and Ki ) 0.071 µM).10 Its
inhibitory action appears to involve the acyl transfer of
the anhydride to the hydroxyl group of serine-195,
generating a stable acyl-enzyme intermediate. The
anhydride structure is important for the activation of
carbonyl reactivity of 1, since 2,2-dimethyl-3-(N-(4-
cyanobenzoyl)amino)-5-phenylpentanoic acid, which is
an acid form of 1, shows no inhibitory activity for
R-chymotrypsin. 1 possesses a bulky anhydride struc-
ture with dimethyl groups on two symmetric R-carbons.
This may contribute to the stability against hydrolysis
of the anhydride structure in a buffer solution such as
Tris-HCl (pH 8.1). However, anhydride still seems to
be too reactive to use in in vivo studies.
Serine proteases play important roles in numerous
physiological processes, and some of them are involved
in pathological processes. The development of effective
and selective low-molecular-weight synthetic inhibitors
for these proteases has been the focus of several studies
of enzyme mechanisms, in vivo biochemical investiga-
tions, and development of potential therapeutic drugs.1,2
Chymotrypsin-like serine proteases are implicated in a
wide variety of pathological states including inflamma-
tion for cathepsin G3,4 and cardiomyopathy for mast cell
chymase.5 Bovine R-chymotrypsin (EC 3.4.21.1) consti-
tutes a model for the evaluation of new synthetic
inhibitors directed against chymotrypsin-like serine
proteases and a comprehensive approach to elucidate
the biological roles of such targeted proteases. Many
types of inhibitors such as substrate inhibitors, transi-
tion-state inhibitors, affinity labels, active-site alkylat-
ing agents, and mechanism-based enzyme inactivators
have been developed for effective and selective inhibition
of serine proteases.1,2,6-9
Recently, we reported that N-modified trisubstituted
â-amino acid anhydride 1 [2,2-dimethyl-3-(N-(4-cy-
* Corresponding author: Yoshio Hayashi, Ph.D. Phone: +81-44-
797-1313. Fax: +81-44-752-6351. E-mail: yhayashi@Lab1.nsc.co.jp.
To improve the in vivo stability, i.e., to prevent
10.1021/jm980562h CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/05/1999

A New Inhibitor of R-Chymotrypsin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 313
Sch em e 1a
nonspecific acylation toward nontargeted proteins or
self-hydrolysis under physiological conditions, it is
necessary that the electron density at the carbonyl
carbon of the reactive center of the inhibitor is in-
creased. Such increase in electron density, however, will
decrease the reactivity of the carbonyl carbon with the
active-site serine residue, resulting in a reduced rate
of formation of the intermediate complex of the inhibitor
and enzyme. One way to overcome this negative effect
is to incorporate the acyl and leaving group moiety that
interact with the respective binding sites more favorably
into the molecule.11 In particular, such interactions with
the leaving group binding sites are known to increase
the rate of hydrolysis of the normal substrate.12 It is,
therefore, likely that these interactions will also in-
crease the rate of formation of the acyl-enzyme. In
addition, a favorable interaction of the acyl moiety with
its binding site in proteases will also compensate for the
reduced rate of acyl-enzyme formation.
A trisubstituted â-amino acid in anhydride 1 is not a
native amino acid, which has a dimethyl group on the
R-carbon and another substituent on the â-carbon.
These trisubstituted â-amino acids were originally used
for the development of fibrinogen receptor antagonists
and found to be effective in restricting the conforma-
tional mobility by steric repulsion.13,14 In anhydride 1,
two trisubstituted â-amino acids will also restrict the
free rotation of the backbone of the molecule and fix the
conformation, which probably enables the molecule to
interact with enzymes with a higher affinity. Therefore,
we have synthesized an extensive series of compounds
containing an R,R-dimethyl-â-alkyl-â-amino acid deriva-
tive and a leaving group with modified reactivity such
as esters or amides instead of the anhydride structure.
In such modifications, one of the phenylalanine
residue-inserted derivatives, N-[2,2-dimethyl-3-(N-(4-
cyanobenzoyl)amino)-5-phenylpentanoyl]-L-phenylala-
nine ethyl ester (2), exhibited a relatively strong inhibi-
tory activity. We modified this compound and found a
new potent inhibitor for chymotrypsin-like serine pro-
teases, N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)n-
onanoyl]-L-phenylalanine ethyl ester (3), which exhibits
anti-R-chymotrypsin activity comparable to that of
anhydride 1.
a
(a) 4-CN-Bz-OSu, Bz-Cl, or (Boc)2O, Et3N, DMF; (b) H-Phe-
OEt, -OMe, -O-n-Pr, -O-i-Pr, -OBzl, or -O-t-Bu, WSCD‚HCl, HOBt,
CH2Cl2; (c) 4 N HCl-dioxane, 0 °C; (d) aryl-COOH, HOBt,
WSCD‚HCl, CH2Cl2.
In this study, we report a stable ester-type potent
inhibitor of chymotrypsin-like proteases and a compre-
hensive investigation of the structural factors necessary
for its potent activity and the kinetics of inhibition of
R-chymotrypsin by 3.
Ch em istr y
Inhibitor 3 and its analogues were generally synthe-
sized by the method shown in Scheme 1 as a diastere-
omeric mixture (a 50:50 mixture of RS and SS). In
Scheme 1, â-substituted-R,R-dimethyl-â-alanine 414 was
coupled with the N-hydroxysuccinimide ester of 4-cy-
anobenzoic acid, benzoyl chloride, or (Boc)2O in the
presence of triethylamine to yield the corresponding
amides or N-Boc-â-substituted-R,R-dimethyl-â-alanine
5, which was coupled with phenylalanine ethyl ester or
corresponding other esters such as methyl, n-propyl,
isopropyl, benzyl, and tert-butyl using 1-ethyl-3-
(3-(dimethylamino)propyl)carbodiimide hydrochloride
(WSCD‚HCl)15 in the presence of 1-hydroxybenzotriazole
(HOBt)16 to yield the target compounds. The 4-cy-
anophenyl-modified or -substituted analogues 17-20
were synthesized from 21 by the deprotection of the
t-Boc group of 21 by 4 N HCl-dioxane, followed by

314 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Ta ble 1. Structure-Activity Relationships of the Analogues of 3
Iijima et al.
IC50 (µM)a
R-chymotrypsin
compd
R1
R2
R3
trypsin
stabilityb (%)
1
2
3
6
7
nac
na
na
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
0.0021
0.25
0.0026
3.4
ni
2.2
27
ni
ni
ni
8.6
ni
4.2
17
0.30
nif
0.54
ni
ni
60
ni
ni
ni
ni
ni
ndd
nd
>99
91
11
90
89
41
3
<1
61
16
nd
<1
nd
nd
6
nd
nd
98
97
98
nd
nd
4-CN-Phe
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
Ph
2-phenylethyl
n-hexyl (C6)g
Et (C2)
n-propyl (C3)
isopropyl (C3)
n-butyl (C4)
isobutyl (C4)
n-pentylh (C5)
n-heptyl (C7)
n-octyl (C8)
n-decyl (C10)
n-dodecyl (C12)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
n-hexyl (C6)
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
ni
220
560
ni
ni
ni
ni
6.3
ni
17
6.7
ni
3,4-diCl-Phi
3,4-diF-Phj
2-F-4-CN-Phk
2-furyl
tert-butoxy
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
4-CN-Ph
7.0
1.5
ni
12
1.0
1.1
0.23
0.086
230
73
n-propyl
isopropyl
benzyl
tert-butyl
ni
a
Inhibition of bovine R-chymotrypsin and bovine trypsin. The IC50 values of chymostatin for R-chymotrypsin and leupeptin for trypsin
are 0.02 and 0.44 µM, respectively. All experiments were performed in triplicate. Standard deviations are less than 15% of the mean
values with the exception of compounds 1, 6, 16, 21, and 22 (29%, 19%, 17%, 18%, and 31% in R-chymotrypsin assay, respectively).
b
d
Percentages of intact compounds after R-chymotryptic cleavage at 37 °C for 120 min. c na, not applicable. nd, not determined. e 4-CN-
Ph, 4-cyanophenyl. f ni, no inhibition at 1 mM. g Number of carbon atoms at R2 position. normal-Pentyl. 3,4-diCl-Ph, 3,4-dichlorophenyl.
h
i
j
k
3,4-diF-Ph, 3,4-difluorophenyl. 2-F-4-CN-Ph, 2-fluoro-4-cyanophenyl.
Ta ble 2. Kinetic Parameters for Inactivation of Bovine
R-Chymotrypsin by Inhibitor 3 and Its Ester Analogues
coupling with 3,4-dichlorobenzoic acid, 3,4-difluoroben-
zoic acid, 2-fluoro-4-cyanobenzoic acid, and 2-furoic acid
for 17-20, respectively, using the above WSCD-HOBt
procedure. Purification was carried out using silica gel
column chromatography.
a
b
ester
form
Kia
(µM)
kinact
kinact/Ki
IC50
compd
(s-1
)
(M-1 s-1
)
(µM)
3
22
23
24
ethyl
methyl
n-propyl
isopropyl
0.0045
12
0.34
0.070
0.0028
0.020
0.0057
0.0035
630000
1600
17000
51000
0.0026
1.1
0.23
Resu lts
0.086
a
The progress curves of the substrate hydrolysis were analyzed
by the zero-time method described by Hart and O’Brien.18 The
concentration of inhibitors used here were 0.1 µM for compound
3, 10 µM for compound 22, 1 µM for compound 23, and 0.33 µM
Str u ctu r e-Activity Rela tion sh ip Stu dies of An a -
logu es of 3. As shown in Table 1, 3 inhibited both
R-chymotrypsin and trypsin with an IC50 of 2.6 and 540
nM, respectively, indicating that this ester is about 200
times more selective against R-chymotrypsin than against
trypsin, and these activities were almost comparable to
those of anhydride inhibitor 1 (IC50 ) 2.1 and 300 nM,
respectively). Since 3 is a mixture of two diastereomers
due to the existence of an asymmetric carbon atom at
the â-position, in addition to the L-phenylalanine resi-
due, both diastereomers (SS and RS) were separated
by normal phase HPLC, and their inhibitory activities
against chymotrypsin-like serine proteases were mea-
sured. As shown in Table 3, both diastereomers exhib-
ited almost the same inhibitory activities against those
proteases, indicating that the potent inhibition of 3 is
nonenantioselective at the â-position.
b
for compound 24. See Table 1.
tion of the R1 position (4-cyanophenyl group), deletion
of the cyano group (16) resulted in a 6500 time decrease
in the inhibitory activity against R-chymotrypsin com-
pared with that of 3. Introduction of halogen atoms (17
and 18) or substitutions of the 4-cyanophenyl group with
a 2-furyl or tert-butoxy group (20 and 21) also decreased
the activity. Moreover, introduction of a fluorine atom
at the 2-position of the 4-cyanophenyl group (19)
resulted in total loss of the activity. Although we
extensively modified the same part in 2 with substitu-
ents such as 4-halophenyl, 4-nitrophenyl, 4-(methoxy-
carbonyl)phenyl, and 4-(aminosulfonyl)phenyl, these
analogues were more than 2000 times less active than
2 (data not shown). These results indicate that the
4-cyanobenzoyl group is one of the best substituents in
the R1 position for a potent anti-R-chymotryptic activity.
The R2 position (â-substituent) was also modified with
To elucidate the structural factors responsible for the
potent activity of 3, we focused on three parts of the
molecule, the 4-cyanophenyl group, the â-substituent at
the â-amino acid residue, and the ester structure, and
synthesized a series of analogues of 3. In the modifica-

A New Inhibitor of R-Chymotrypsin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 315
Ta ble 3. Inhibitory Activities of 3 and Its Analogues against Serine Proteases
IC50 (µM)a
chymase
compd
R2
R3
ethyl
ethyl
ethyl
methyl
n-propyl
isopropyl
ethyl
ethyl
ethyl
R-chymotrypsin
cathepsin G
elastase
3
n-hexyl
n-hexyl
n-hexyl
n-hexyl
n-hexyl
n-hexyl
n-butyl
n-pentyl
n-heptyl
n-octyl
0.0026
0.0016
0.0051
1.1
0.44
0.22
0.36
19
3.2
0.15
510
ni
0.0087
0.0084
0.010
1.5
0.20
0.077
83
ni
440
6.6
0.0070
0.0086
0.014
4.2
0.27
0.20
710
ni
ni
2.7
3-F b
3-Rc
22
23
24
9
0.23
0.086
27
11
12
13
nid
ni
8.6
ni
62
ethyl
a
Inhibition of bovine R-chymotrypsin, human chymase, human cathepsin G, and porcine elastase. The IC50 values of chymostatin for
R-chymotrypsin, chymase, and cathepsin G and elastatinal for elastase are 0.02, 0.44, 0.025, and 0.22 µM, respectively. All experiments
were performed in triplicate. Standard deviations (SEM) are less than 20% of the mean values with the exception of compound 22 (R-
chymotrypsin), 13 (chymase), and 13 (elastase), in which those SEM values are 31%, 21%, and 21% of the mean values, respectively.
b
3-F , a diastereomer of 3, which was eluted as a front peak by normal phase HPLC. c 3-R, a diastereomer of 3, which was eluted as a
d
rear peak by normal phase HPLC. ni, no inhibition at 1 mM.
alkyl groups of different lengths. The inhibitory activity
was observed in analogues bearing the linear alkyl
chains, whose carbon number was even, such as C2, C4,
C6, and C8 (6, 9, 3, and 13). Maximum inhibition was
noted after the introduction of a n-hexyl group (3), and
compounds with shorter or larger alkyl chains were less
potent. However, analogues with linear alkyl chains,
whose carbon number was odd, such as C3, C5, and C7
(7, 11, and 12), showed no inhibitory activity. We also
evaluated the inhibitory activity of the analogues with
the branched alkyl groups such as isopropyl 8 and
isobutyl 10. The isopropyl analogue 8 containing odd-
numbered (three) carbon atoms showed an inhibitory
activity with an IC50 value of 2.2 µM, which was
comparable to that of the ethyl analogue 6 (3.4 µM),
although the n-propyl analogue 5 showed no inhibitory
activity. Isobutyl analogue 10 containing even-num-
bered (four) carbon atoms showed no activity similar to
the n-propyl analogue 7 (no activity), although the
n-butyl analogue 9 showed considerable anti-chymot-
ryptic activity. These results imply that the inhibitory
activity of analogues with the branched alkyl groups is
dependent on the length of their main chains, not the
total number of carbon atoms.
motrypsin by analyzing the formation of degraded
products, especially the corresponding free acid. As
shown in Table 1, analogues of 3 bearing linear alkyl
chains, whose carbon numbers were odd, such as C3,
C5, and C7 (7, 11 and 12), were effectively hydrolyzed
by the protease, yielding only the corresponding acids.
However, those analogues bearing the linear alkyl
chains, whose carbon numbers were even, such as C2,
C4, and C6 (6, 9, and 3), strongly resisted hydrolysis to
the corresponding acids by the protease, except C8 (13)
and C10 (14) in which 40% and 85% of the acid
formation was observed during 2 h of incubation,
respectively. In particular, compound 3 with a linear
alkyl chain of C6 was rarely hydrolyzed during this
period (less than 1%) and even less so in the 3-h
incubation (not shown). Although these analogues con-
tain one asymmetric carbon atom at the â-position in
addition to the L-phenylalanine residue, i.e., mixture of
two diastereomers, both diastereomers, whose corre-
sponding acids can be detected separately by HPLC,
showed almost the same behavior against chymotryptic
cleavage. (The percent values of stability shown in Table
1 are the average of both diastereomers, and each
deviation was less than 1% with the exception of
compounds 6 and 7 with a deviation of 5% and 12%,
respectively.) These results indicate that the recognition
of compounds as substrates by R-chymotrypsin is non-
enantioselective at the â-position.
In modification of the R3 position (ester), compounds
with alkyl groups such as methyl, n-propyl, and isopro-
pyl groups (22-24) were 30-400 times less active than
3, and compounds with the benzyl and tert-butyl groups
(25 and 26) showed very weak activity, indicating that
the ethyl ester in this position is one of the best
functional groups associated with the potent anti-R-
chymotryptic activity.
Ch ym otr yp tic Clea va ge of An a logu es of 3. The
mechanism underlying the marked change in the in-
hibitory activity observed with only slight modifications
of the structure, including the 4-cyanophenyl group, the
side chain at the â-position, or the ester, is not clear.
One possible explanation is that some of the analogues
may be cleaved by the enzyme as a substrate, resulting
in loss of activity. To investigate such a possibility, we
examined the stability of each compound against R-chy-
The relationship between anti-R-chymotryptic activity
and stability against chymotryptic cleavage is plotted
in Figure 1. As this figure shows, anti-R-chymotryptic
activities clearly correlated with the stability against
chymotrypsin, suggesting that the reduced inhibitory
activities observed when the length of the alkyl chains
was varied were largely dependent on the instability of
the acyl-enzyme. This is also true with compounds with
the branched alkyl chains such as isopropyl and isobutyl
and compounds with relatively long linear alkyl chains
such as C8 (13) and C10 (14) at the â-position.
As shown in Table 1, compounds with the cyano group
deleted and the fluorine atom added (16 and 19) were

316 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Iijima et al.
F igu r e 2. Reactivation of chymotrypsin by the addition of
hydrazine. Chymotrypsin (1.25 µg/mL) was inactivated by
treatment with 5 µM compound 3 for 20 min at room temper-
ature and was dialyzed overnight against HEPES buffer (pH
7.4) containing 100 mM NaCl at 4 °C. Hydrazine solution was
added to the dialysate (final concentration ) 0.6 M) followed
by incubation at room temperature. The activity of reactivated
chymotrypsin was determined by adding a chromogenic sub-
strate (final concentration ) 750 µM). R-Chymotrypsin without
incubation with compound 3 was used as a control, and the
chymotryptic activity of each sample is expressed as percent-
age of the control.
F igu r e 1. Relationship between IC50 values and stability. The
IC50 value of each compound was plotted against its stability
against chymotryptic cleavage. The number of carbon atoms
is indicated near the symbol.
effectively hydrolyzed to the corresponding acids, indi-
cating that the 4-cyanophenyl group was also an im-
portant unit for resisting chymotryptic cleavage. Since
these modifications resulted in a reduction of anti-R-
chymotryptic activities, the reduced activities will also
be largely dependent on the instability of the acyl-
enzyme.
We also examined the effects of ester structures on
chymotryptic cleavage. Formation of the corresponding
acids was 2.4% (methyl), 3.1% (n-propyl), and 2.7%
(isopropyl) after incubation with chymotrypsin for 2 h,
respectively, which were comparable to that of 3 (ethyl,
<1%).
Com p etitive In h ibition Stu d ies of 3. In the next
step, we investigated the kinetics of inhibition of R-chy-
motrypsin by 3 using a chromogenic substrate, N-
succinyl-Ala-Ala-Pro-Phe-pNA.17 In competitive binding
assays, inhibitor 3 appeared to be an active-site-directed
reagent. 3 behaved as a competitive inhibitor of R-chy-
motrypsin. The initial Ki value for 3, determined from
the double-reciprocal plot (see Figure 3, Supporting
Information), was 13.6 µM.
forms, and the inhibitory activity of each compound was
dependent on Ki values.
Recover y of En zym e Activity by Hyd r a zin e. The
kinetic studies showed that compound 3 inhibited
R-chymotrypsin in a competitive manner, suggesting
that 3 may interact with the active site of the enzyme
and form a stable acyl-enzyme. To examine this pos-
sibility, the reactivation kinetics of the inactivated
enzyme was studied. R-Chymotrypsin, fully inactivated
by treatment with 3, was dialyzed at 4 °C overnight
against HEPES buffer (pH 7.4), and the chymotryptic
activity was determined by the addition of the chro-
mogenic substrate. As shown in Figure 2, the hydrolytic
activity was not recovered even after dialysis, and
additional incubation at room temperature for 24 h also
failed to reactivate the enzyme, indicating that the
deacylation rate constant of 3 is too small to be
determined. These results indicated that the inhibition
was irreversible. However, reactivation of the inacti-
vated enzyme occurred when the enzyme was treated
with buffered hydrazine. Although the reactivation rate
was not very fast, about 70% of chymotryptic activity
was recovered after a 24-h incubation. These results
suggest that compound 3 inhibits R-chymotrypsin by
forming an irreversible acyl-enzyme, probably by react-
ing with the hydroxyl group of the active site Ser residue
(195) or with the imidazole ring of the His residue (57).
In h ibition Kin etics of 3 a n d Its Ester An a logu es.
As shown in Figure 4 (Supporting Information), inhibi-
tion of R-chymotrypsin by inhibitor 3 was time-depend-
ent and the hydrolysis rate of the substrate did not
reach a steady state at the end of the experimental
period (300 s). Therefore, Ki (final) and kinact values were
calculated by the progress curve method.18 The method
used to calculate these values is described in the
Experimental Section. Using a semilogarithmic plot of
velocity-time, our analysis showed that the rate of
substrate hydrolysis was linear (data not shown). Using
this relationship, the estimated Ki and kinact values were
0.0045 µM and 0.0028 s-1, respectively, resulting in a
In h ibition of Oth er Ser in e P r otea ses. The IC50
values of inhibitor 3 and its analogues against several
serine proteases are shown in Table 3. 3 inhibited
cathepsin G and elastase at nanomolar concentrations,
and the inhibitory activity against human chymase was
less potent. We also examined the effects of modification
of both the â-position and the ester structure and found
that the profiles of inhibitory potency against cathepsin
G, elastase, and chymase were almost similar to those
against R-chymotrypsin.
k
inact/Ki of 630 000 M-1 s-1. Table 2 shows the kinetic
constants including Ki, kinact, and kinact/Ki values, cal-
culated by the same method, for inhibition of R-chymot-
rypsin by inhibitor 3 and its ester analogues. The Ki
values, not kinact, were largely affected by the ester

A New Inhibitor of R-Chymotrypsin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 317
Discu ssion
pounds were hydrolyzed to the free acid, suggesting that
the acyl-enzyme is formed but the deacylation rate is
very high. In contrast, in compounds with an alkyl chain
and even carbon numbers, the acyl-enzyme was stable
and the deacylation rate was very slow. The mechanism
that determines the stability of the acyl-enzyme is not
known. Baek et al.19 discussed the factors that affect
the deacylation rate of the acyl-enzyme in their study
on alternate substrate inhibitors with an enol lactone
structure. These factors are (a) a reactivity of ester in
the acyl-enzyme,20-22 (b) a steric blockade or ionic
repulsion of the attacking water molecules,23-26 (c) a
conformational demand that pulls the acyl-enzyme
carbonyl oxygen out of the oxyanion hole, a site where
the reactivity with nucleophiles is thought to be en-
hanced,27,28 and (d) a conformational alternation of other
portions of the catalytic machinery of the enzyme such
as His-57 moving from the active “in” position to the
“out” position.29 In the case of the compounds presented
here, it is probable that the catalytic inertness derived
from mechanisms such as factors b-d may be dependent
on the alternate positioning of the compound, caused
by different lengths of the alkyl chains at the â-position,
in the narrow pocket of the active site of the enzyme.
We are about to begin a study of crystallization of 3 and
R-chymotrypsin complex to elucidate the precise binding
mechanism. This approach may provide the possible
answer to explain the effects of the length of the alkyl
chains at the â-position on the inhibitory activity.
The ethyl ester structure is also an interesting feature
of inhibitor 3, since the inhibitory activity was markedly
reduced following the conversion of the structure of this
ester to other similar alkyl esters such as methyl,
n-propyl, isopropyl, and tert-butyl esters, although these
analogues were resistant to enzymatic cleavage. Deter-
mination of the kinetic constants in the inhibition of
R-chymotrypsin by compound 3 and its ester form
derivatives showed that Ki values, not kinact values, were
largely affected by the deviation of the ester form (Table
2). These results suggest that the rate of transition from
the intermediate inhibitor-enzyme complex to the
stable acyl-enzyme may not be dependent to a large
extent on the ester form. The leaving group of the ester
moiety may play at least two opposite roles in these
steps. First, the leaving group may be involved in the
interaction between the compound and the binding sites
on the enzyme, through which the compound binds more
tightly to the enzyme. Second, the leaving group may
donate electrons onto the carbonyl carbon, which re-
duces the reactivity of that carbon with the active site
of the enzyme. Based on our finding of a potent activity
of inhibitor 3 which has an ethyl ester, it is likely that
the ethyl ester interacts strongly with the leaving group
binding sites on the enzyme and overcomes the negative
effects of its electron donation.
In our previous study,10 we developed a series of
inhibitors for chymotrypsin-like enzymes, which possess
an anhydride structure. We demonstrated that one of
the most potent inhibitors, anhydride 1 [2,2-dimethyl-
3-(N-(4-cyanobenzoyl)amino)-5-phenylpentanoic anhy-
dride], inhibited chymotrypsin-like enzymes including
chymotrypsin, chymase, and cathepsin G by competing
with the substrates and irreversibly acylating the active
site of the enzymes. Because of the anhydride structure,
anhydride 1 is very reactive, making nonspecific acy-
lating reactions inevitable in vivo.
In the present study, we report a stable ester-type
inhibitor, N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)-
nonanoyl]-L-phenylalanine ethyl ester (3), which is a
trisubstituted â-amino acid derivative, and its ana-
logues. As shown in Figure 3 (Supporting Information),
inhibitor 3 is a competitive inhibitor of R-chymotrypsin.
Dialysis of the inactivated enzyme showed that the
action of inhibitor 3 is irreversible and reactivation by
treatment with hydrazine suggests the formation of the
acyl-enzyme. Although the exact acylation site is not
known, the Ser residue at the active site of the enzyme
is probably acylated. Inhibition of R-chymotrypsin by
inhibitor 3 is time-dependent, suggesting that it is a
relatively slow binding inhibitor. Analysis of the progress
curve (Figure 4, Supporting Information) resulted in
kinetic constants of kinact ) 0.0028 s-1 and Ki ) 0.0045
µM, yielding a kinact/Ki value of 630 000 M-1 s-1. These
kinetic constants are almost comparable to those of
anhydride inhibitor 1 (kinact/Ki ) 242 000 M-1 s-1, kinact
) 0.017 s-1, and Ki ) 0.071 µM). The ethyl ester
structure in 3 will be more stable under physiological
conditions compared with the anhydride structure, due
to the reduced electrophilicity at the carbonyl carbon,
which is a reactive center and a target of the active-
site Ser-195 in R-chymotrypsin. Because a reduced
reactivity of the reactive center will generally result in
a reduced inhibitory activity by slowing the rate of acyl-
enzyme formation, the potent activity of inhibitor 3
suggests that other structural factors may compensate
the reduced reactivity.
To investigate the structural factors involved in the
interaction between inhibitors and the enzyme, we
conducted a structure-activity relationship (SAR) study
and a structure-stability relationship study among
inhibitor 3 and its analogues. We focused on three parts
of the molecule: the 4-cyanophenyl group, the â-sub-
stituent at the trisubstituted â-amino acid residue, and
the ester structure. Since the phenylalanine residue in
inhibitor 3 is the most typical amino acid residue in
peptide substrates recognized by chymotrypsin-like
enzymes, this residue would interact with the S1 site
in acyl binding sites of R-chymotrypsin. In the present
study, this moiety was not modified.
The 4-cyanophenyl group was also very important
moiety for the potent inhibitory activity. 16, a compound
without the cyano group, and 19, a compound with an
additional fluorine atom at the 2-position of the 4-cy-
anophenyl ring, exhibited a markedly reduced inhibitory
activity as well as stability against enzymatic cleavage.
These results suggest that this cyanophenyl group may
be responsible for the stabilization of the acyl-enzyme.
Since the phenyl group would be located at the P3
Modification of the â-substituent at the trisubstituted
â-amino acid residue largely affected the inhibitory
activity as shown in Table 1. The inhibitory activity
against R-chymotrypsin is dependent on the number of
carbon atoms of the alkyl chains introduced into the
â-position. Introduction of the linear alkyl chain with
an odd carbon number resulted in complete loss of
activity. On the other hand, the structure-stability
relationship study showed that these inactive com-

318 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Iijima et al.
position of peptide substrates, this group may be
implicated in the interaction with the S3 site of the
enzyme. Therefore, it is likely that the 4-cyano group
stabilizes the acyl-enzyme by interacting with a site in
or near the S3 pocket or by modifying the distribution
of the π-electrons on the phenyl ring. Such interaction
may lead to the above-mentioned catalytic inertness
resulting in a reduced deacylation rate of the acyl-
enzyme.
serine proteases and defined the structural factors
necessary for its potent activity. The structural factors
responsible for the potent inhibitory activity of 3 eluci-
dated in the present study can be summarized as
follows: (1) the ethyl ester that functions as a group
enforcing specific acyl-enzyme formation, (2) the n-hexyl
group at the â-position and the 4-cyanophenyl group,
which function as stabilizers for the acyl-enzyme, and
(3) the phenylalanine residue which functions as a
moiety involved in the specific recognition by the
enzyme upon acyl-enzyme formation. 3 is one of the
ideal potent substrate inhibitors with a high binding
affinity and intensely slow deacylation rate. These
results provide an intriguing basis for further develop-
ments in the design of specific serine protease inhibitors.
Small synthetic molecules, such as Bz-Phe-OEt, are
known to be better substrates for R-chymotrypsin with
a relatively higher deacylation rate (5500 min-1).30
However, the deacylation rate of inhibitor 3, which
contains the same Phe-OEt structure, seems to be very
slow, although the exact rate was not determined. These
results also suggest the significance of the n-hexyl group
at the â-position and the 4-cyanophenyl group in the
slow deacylation.
Exp er im en ta l Section
En zym e Assa ys. The inhibitory effects of each compound
on the enzymatic activities of serine proteases were evaluated
using purified enzymes and chromogenic substrates. Enzymes
and their substrates used in the present study were as
follows: N-succinyl-Ala-Ala-Pro-Phe-pNA17 (1.5 mM) for bovine
pancreatic R-chymotrypsin (47 ng/mL, type I-S; Sigma Chemi-
cal Co., St. Louis, MO); N-succinyl-Ala-Ala-Pro-Phe-pNA (3
mM) for human leukocyte cathepsin G (2.5 U/mL; Elastin
Products Co., Inc.); N-succinyl-Ala-Ala-Ala-pNA (1 mM) for
porcine pancreatic elastase (0.8 µg/mL, Sigma type III); Bz-
L-Arg-pNA (1 mM) for porcine pancreatic trypsin (1 U/mL;
Wako Pure Chemicals); N-succinyl-Ala-Ala-Pro-Phe-pNA (1.5
mM) for human recombinant chymase. All experiments were
carried out in 50 mM HEPES buffer (pH 7.4) containing 0.1
mM NaCl, except for the buffer used for chymase, which
contained 50 mM Tris-HCl (pH 8.0) and 1 M KCl. The enzyme
solutions were mixed with substrate solutions in the presence
of various concentrations of each compound and incubated at
37 °C for 120 min, and absorbance at 405 nm was measured
to determine enzymatic activities. The concentrations of
enzymes and substrates mentioned above gave an increment
of the absorbance unit at 405 nm to 1.0 without inhibitors
under our assay conditions. IC50 values were calculated from
the dose-inhibition curves. Chymostatin was used as a control
compound for the inhibition of R-chymotrypsin, cathepsin G,
and chymase. Elastatinal and leupeptin were also used for
elastase and trypsin, respectively. We confirmed that devia-
tions of IC50 values were less than 10% from repetitive
determinations of IC50 values of these control compounds.
Com p etitive In h ibition Assa y of r-Ch ym otr yp sin . The
inhibitory effect of the compounds against chymotrypsin was
estimated from the effects on the initial velocity of hydrolysis
of the substrate; 2 µL of DMSO solution of the inhibitor was
added to 400 µL of 60 nM bovine R-chymotrypsin solubilized
in HEPES buffer (pH 7.4) containing 100 mM NaCl, and
immediately after mixing, 400 µL of the substrate (N-succinyl-
Ala-Ala-Pro-Phe-pNA) solution was added at various concen-
trations. An increase in absorbance at 405 nm was followed
for 30 s at room temperature to determine the initial velocity
of the substrate hydrolysis.
The importance of trisubstituted â-amino acids as a
conformation-restricting linear template was first shown
in our studies on fibrinogen receptor (GPIIb/IIIa) an-
tagonists. In these studies, we confirmed that trisub-
stituted â-amino acids were very useful in restricting
the free rotation of the backbone structure of the
molecules and fixing the molecular conformation. Trisub-
stituted â-amino acids have also shown to be useful for
the introduction of a functional group into the molecule,
by substituting the group at the â-position. On the basis
of these findings, we expected that trisubstituted-â-
amino acids will be generally applicable to the amide
bond-based small molecules for the purpose of fixing the
molecular conformation and the addition of the interac-
tion site. Protease inhibitors are one of the possible
targets, because to develop potent and selective protease
inhibitors, the molecule should have multiple binding
sites recognized by the enzyme and should be tightly
fixed to the appropriate conformation so as to position
these multiple functional groups in a proper way.
Therefore, in the present study we introduced a trisub-
stituted â-amino acid to the inhibitors for fixing the
molecules and introducing the P2 moiety, which is
known as the interaction site recognized by the S2 site
of the enzyme. The present study showed that only a
minor modification, such as the addition or deletion of
a C1 unit at the â-substituent and the introduction of a
substituted group to the phenyl group, resulted in a
major reduction in the inhibitory activity. This hyper-
sensitivity may be attributed to the conformational
rigidity of our compounds, a property thought to be
derived from that of the trisubstituted â-amino acid
introduced into the molecules. Further molecular mod-
eling studies are necessary to demonstrate the signifi-
cance of the trisubstituted â-amino acids as a confor-
mation-restricting unit. These studies are currently
being conducted in our laboratory.
Kin etic Stu d ies by P r ogr ess Cu r ve Meth od . Because
inhibition of R-chymotrypsin by the present compounds was
time-dependent, the kinetics of the inhibition was analyzed
by the progress curve methods described by Hart and O’Brien.18
Briefly, 400 µL of 60 nM bovine R-chymotrypsin was mixed
with 400 µL of chromogenic substrate solution in the presence
of an appropriate concentration of inhibitors, and increased
absorbance at 405 nm was followed for 300 s at 25 °C. Using
The inhibition profiles of inhibitor 3 and its analogues
against several serine proteases including chymot-
rypsin, chymase, cathepsin G, and elastase suggested
a similar structure of the active sites, recognized by this
series of compounds, in these proteases and the presence
of a common inhibitory mechanism.
a
semilogarithmic plot, the velocity of hydrolysis of the
substrate (v) at each time point was plotted as a function of
time, which gave a linear regression line. Ki is calculated by
using the following equation:
In conclusion, we synthesized a novel potent inhibitor,
N-[2,2-dimethyl-3-(N-(4-cyanobenzoyl)amino)nonanyl]-
L-phenylalanine ethyl ester (3), for chymotrypsin-like
Ki ) Km[I]/((Km + [S])(vc/v0 - 1))

A New Inhibitor of R-Chymotrypsin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 319
where Km is the Michaelis constant (41 µM; determined by the
standard method), [ I ] is the inhibitor concentration, [S] is
the substrate concentration (200 µM), vc is the velocity of a
control reaction carried out in the absence of inhibitor, and v0
is the velocity of the reaction at time zero, which was
determined from the y-intercept of the time-ln v plot. kinact is
also obtained from the equation:
trimethylsilylimine via a cannula at a rate such that the
temperature did not exceed -50 °C. The mixture was stirred
at -70 °C for 60 min, allowed to warm to room temperature,
and stirred for an additional 18 h. In the next step, 100 mL of
saturated aqueous NH4Cl was added to the reaction mixture,
and the mixture was extracted three times with 50 mL of
diethyl ether. The combined organic layers were washed with
saturated NaCl, dried over Na2SO4, and concentrated in vacuo.
The residual pale-yellow oil was applied to a silica gel column
(5 × 50 cm) and eluted with EtOAc-hexane (1:4) to give 4.78
g (52%) of corresponding azetidinone as a pale-yellow oil: 1H
NMR (270 MHz, CDCl3) δ 0.90 (t, J ) 8.4 Hz, 3H), 1.17 (s,
3H), 1.31 (s, 3H), 1.21-1.40 (m, 8H), 1.42-1.64 (m, 2H), 3.29
(dd, J ) 5.9, 8.3 Hz, 1H), 5.91 (br s, 1H, NH); MS (ESI) m/z
184 (M + H)+, 206 (M + Na)+. A mixture of this azetidinone
(1.0 g, 5.5 mmol) and 6 N HCl (100 mL) was stirred at room
temperature for 48 h. The solution was condensed under
reduced pressure, and then toluene (30 mL) was added to the
residue and evaporated twice to give 1.28 g (98%) of a
hydrochloride salt of 4-n-hexyl-3,3-dimethyl-2-azetidinone (4b)
as a white solid: mp 150-152 °C; MS (ESI) m/z 202 (M + H)+.
Anal. (C11H23NO2‚0.9HCl) C, H, N.
Compounds 4a , c-l were prepared according to procedure
A described for preparation of 4b. See ref 14 for the syntheses
and physicochemical data for compounds 4a -h .
3-Am in o-2,2-d im eth yld eca n oic a cid (4i) (h yd r och lo-
r id e): 69% yield from ethyl isobutyrate; white solid; mp 159-
161 °C; MS (ESI) m/z 216 (M + H)+. Anal. (C12H25NO2‚HCl)
C, H, N.
3-Am in o-2,2-d im eth ylu n d eca n oic a cid (4j) (h yd r och lo-
r id e): 66% yield from ethyl isobutyrate; white solid; mp 158-
160 °C; MS (ESI) m/z 230 (M + H)+. Anal. (C13H27NO2‚HCl‚
0.25H2O) C, H, N.
3-Am in o-2,2-d im et h ylt r id eca n oic a cid (4k ) (h yd r o-
ch lor id e): 11% yield from ethyl isobutyrate; white solid; mp
141-143 °C; MS (ESI) m/z 258 (M + H)+. Anal. (C15H31NO2‚
HCl) C, H, N.
kinact ) (∆ln v/∆t)(Ki/([I](1 - R)) + 1)
where (∆ln v/∆t) is the slope of the regression line in the
semilogarithmic plot mentioned above, R ) [S]/(Km + [S]), and
Ki is the calculated dissociation constant.
Sta bility of In h ibitor 3 An a logu es a ga in st Ch ym ot-
r yp tic Clea va ge. Ten microliters of 5 mM inhibitor solution
was mixed with 990 µL of 625 ng/mL bovine R-chymotrypsin
solution. After incubation of the mixture at 37 °C for 120 min,
an aliquot of the sample was applied to HPLC, and the intact
compound and cleaved free acid were determined. HPLC was
carried out using a J asco system (800 series) equipped with a
UV/vis detector and an integrator. The solvent system used
for analytical HPLC consisted of a binary system, water
containing 0.1% TFA, and acetonitrile containing the same
TFA as the organic modifier. The dimensions of the column
used for analytical chromatography were 4.6 × 250 mm
(Wakosil-II 5C18 HG). The analytical conditions were a linear
gradient with 50% to 75% acetonitrile in 0.1% TFA over 25
min at a flow rate of 1.0 mL/min. Stability was expressed as
percent of the intact compound remaining in the sample
relative to the initial amount.
Recover y of En zym e Activity by Hyd r a zin e. R-Chy-
motrypsin (1.25 µg/mL) was incubated with 5 µM 3 for 20 min
at room temperature and dialyzed against HEPES buffer
(containing 0.1 M NaCl, pH 7.4) overnight. Hydrazine solution
(HEPES buffer solution, pH 7.4, final concentration was 0.6
M) was added to the dialysates, and enzyme solutions were
incubated at room temperature. An equal volume of the
enzyme solution and the substrate solution (final concentration
was 750 µM) were mixed, and the increase in absorbance at
405 nm was monitored. R-Chymotrypsin solution without
incubation with 3 was used as a control, and chymotryptic
activity of each group was expressed as percent of control.
3-Am in o-2,2-d im eth ylp en ta d eca n oic a cid (4l) (h yd r o-
ch lor id e): 11% yield from ethyl isobutyrate; white solid; mp
137-139 °C; MS (ESI) m/z 286 (M + H)+.
Gen er a l P r oced u r e B for P r ep a r a tion of 5. 3-(N-(4-
Cya n oben zoyl)a m in o)-2,2-d im eth yln a n oic Acid (5b). To
a solution of 4-n-hexyl-3,3-dimethyl-2-azetidinone hydrochlo-
ride (4b) (1.0 g, 4.2 mmol) in DMF (40 mL) were added
triethylamine (1.3 mL, 9.3 mmol) and N-succinimidyl 4-cy-
anobenzoate (1.2 g, 5.0 mmol) at 0 °C, and the resulting
mixture was stirred at room temperature for 18 h. The solvent
was removed by evaporation, and the residue was dissolved
in EtOAc (50 mL). The organic phase was washed with 5%
citric acid and saturated NaCl, dried over Na2SO4 and con-
centrated in vacuo. The residual crude powder (1.45 g) was
applied to a silica gel column (2 × 17 cm) and eluted with
EtOAc-hexane (1:3) to give 0.92 g (66%) of 5b as a white
solid: mp 142-144 °C; MS (ESI) m/z 331 (M + H)+, 353 (M +
Na)+. Anal. (C19H26N2O3‚0.125H2O) C, H, N.
Compounds 5a ,c-n were prepared according to procedure
B described for the preparation of 5b starting from the
appropriate intermediates 4. In the preparation of 5m ,n ,
benzoyl chloride and (Boc)2O were used instead of N-succin-
imidyl 4-cyanobenzoate, respectively. See ref 14 for the
syntheses and physicochemical data for compounds 5a ,c-h .
3-(N-(4-Cya n ob en zoyl)a m in o)-2,2-d im et h yld eca n oic
a cid (5i): 75% yield from 4i; white solid; mp 119-120 °C; MS
(ESI) m/z 343 (M - H)+. Anal. (C20H28N2O3) C, H, N.
3-(N-(4-Cya n oben zoyl)a m in o)-2,2-d im eth ylu n d eca n o-
ic a cid (5j): 70% yield from 4j; white solid; mp 96-97 °C; MS
(ESI) m/z 357 (M - H)+. Anal. (C21H30N2O3‚0.125H2O) C, H,
N.
1
Ch em istr y. H NMR and 13C NMR spectra were recorded
on a J EOL GSX270J spectrometer. The spectra were recorded
with tetramethylsilane (δ ) 0.0 for 1H), CD3OD (δ ) 49.8 for
13C), or CDCl3 (δ ) 77.0 for 13C) as internal reference. Mass
spectra (electrospray ionization, methanol as the mobile phase)
were analyzed with a Finnigan SSQ 7000 spectrometer. High-
resolution fast atom bombardment mass spectra were analyzed
with a J EOL J MS-DX303 spectrometer. A silica gel column
chromatography was performed using Merck 70-230 mesh
silica gel 60. Normal phase HPLC on a semipreparative scale
for the separation of diastereomers of 3 was performed with a
column (Merck, LiChrospher Si 60, 10 × 250 mm, 10 µm)
employing the solvent system with AcOEt-hexane (2:3) at a
flow rate of 4 mL/min on a Waters system (600E series).
Gen er al P r ocedu r e A for P r epar ation of â-Su bstitu ted-
r,r-dim eth yl-â-alan in es. 3-Am in o-2,2-dim eth yln an oic Acid
(4b) (Hyd r och lor id e). To 14 mL (66 mmol) of 1,1,1,3,3,3-
hexamethyldisilazane in 20 mL of dry tetrahydrofuran was
added 40 mL (66 mmol) of 1.65 M n-butyllithium in hexane
at 0 °C over a 10-min period. The mixture was stirred at 0 °C
for 20 min, and the solvent was removed in vacuo until a white
precipitate appeared. To the resulting slurry was added
dropwise a solution of 7.7 mL (55 mmol) of n-heptanal in 15
mL of tetrahydrofuran at -20 °C over a 5-min period. The
resulting solution of trimethylsilylimine was used directly in
the following reaction. To 9.1 mL (65 mmol) of diisopropy-
lamine in 30 mL of tetrahydrofuran was added 36.4 mL (60
mmol) of 1.65 M n-butyllithium in hexane at -70 °C. The
mixture was stirred at -70 °C for 20 min, followed by addition
of 6.7 mL (50 mmol) of ethyl isobutyrate in 10 mL of
tetrahydrofuran over a 10-min period. The solution was stirred
at -70 °C for 60 min, followed by addition of the solution of
3-(N-(4-Cya n oben zoyl)a m in o)-2,2-d im eth yltr id eca n o-
ic a cid (5k ): 81% yield from 4k ; white solid; mp 132-133
°C; MS (ESI) m/z 409 (M + Na)+. Anal. (C23H34N2O3) C, H, N.
3-(N-(4-Cya n ob en zoyl)a m in o)-2,2-d im et h ylp en t a d e-
ca n oic a cid (5l): 66% yield from 4l; colorless oil; MS (ESI)
m/z 413 (M - H)-. Anal. (C25H38N2O3‚0.25H2O) C, H, N.

320 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Iijima et al.
3-(N-Ben zoylam in o)-2,2-dim eth yln an oic acid (5m ): 54%
yield from 4b; white solid; mp 140-142 °C; MS (ESI) m/z 306
(M + H)+. Anal. (C18H27NO3) C, H, N.
3-(N-(Bu t yloxyca r b on yl)a m in o)-2,2-d im et h yln a n oic
a cid (5n ): 82% yield from 4b; white solid; mp 120-122 °C;
MS (ESI) m/z 302 (M + H)+. Anal. (C16H31NO4‚0.75H2O) C,
H, N.
N-[2,2-Dim eth yl-3-(N-(4-cyan oben zoyl)am in o)h exan oyl]-
L-p h en yla la n in e eth yl ester (7): 73% yield from 5d ; white
foam; 1H NMR (270 MHz, CDCl3) δ 0.877 (t, J ) 7.3 Hz, 1.5H),
0.882 (t, J ) 7.3 Hz, 1.5H), 1.19 (s, 3H), 1.22 (s, 3H), 1.04-
1.42 (m, 3H), 1.30 (t, J ) 6.8 Hz, 1.5H),), 1.31 (t, J ) 6.8 Hz,
1.5H), 1.44-1.63 (m, 1H), 3.08-3.26 (m, 2H), 3.94-4.06 (m,
1H), 4.23 (q, J ) 6.8 Hz, 2H), 4.78-4.92 (m, 1H), 6.11 (d, J )
7.8 Hz, 0.5H, NH), 6.20 (d, J ) 7.3 Hz, 0.5H, NH), 7.07-7.15
(m, 2H), 7.18-7.34 (m, 3H), 7.70 (d, J ) 8.3 Hz, 1H), 7.74 (d,
J ) 8.3 Hz, 1H), 7.82 (d, J ) 9.8 Hz, 0.5H, NH), 7.88 (d, J )
8.3 Hz, 1H), 7.94 (d, J ) 8.3 Hz, 1H), 7.88-7.97 (m, 0.5H, NH);
13C NMR (67.5 MHz, CDCl3) δ 13.91, 13.94, 14.1, 14.2, 19.7,
19.8, 22.9, 23.0, 25.1, 25.8, 33.0, 33.2, 37.5, 37.6, 45.1, 45.3,
52.66, 52.72, 57.3, 57.4, 61.8, 61.9, 114.68, 114.74, 118.2, 127.3,
127.4, 127.7, 128.6, 128.7, 129.0, 129.2, 132.3, 132.4, 135.4,
138.3, 138.5, 165.1, 165.3, 171.2, 171.8, 176.6, 177.1; MS (ESI)
m/z 464 (M + H)+, 486 (M + Na)+. Anal. (C27H33N3O4) C, H,
N.
Gen er a l P r oced u r e C for P r ep a r a tion of Ta r get Com -
p ou n d s. N-[2,2-Dim eth yl-3-(N-(4-cya n oben zoyl)a m in o)n -
on a n oyl]-L-p h en yla la n in e Eth yl Ester (3). To a solution
of 5b (0.99 g, 3.0 mmol) and L-phenylalanine ethyl ester
hydrochloride (0.92 g, 4.0 mmol) in CH2Cl2 (30 mL) were added
1-hydroxybenzotriazole (HOBt; 0.55 g, 3.6 mmol) and 1-ethyl-
3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (WSCD‚
HCl; 0.75 g, 3.9 mmol) at 0 °C, and the mixture was stirred at
room temperature for 18 h. After the solvent was removed in
vacuo, the residue was dissolved in EtOAc (50 mL) and washed
with 5% citric acid, 5% sodium bicarbonate, and saturated
NaCl, dried over Na2SO4, and concentrated in vacuo. The
residual oil was applied to a silica gel column (2.2 × 20 cm)
and eluted with EtOAc-hexane (1:3 to 1:2) to give 1.47 g (75%)
of 3 as a colorless oil: 75% yield from 5b; white foam; 1H NMR
(270 MHz, CDCl3) δ 0.83 (t, J ) 6.8 Hz, 1.5H), 0.85 (t, J ) 6.8
Hz, 1.5H), 1.19 (s, 3H), 1.22 (s, 3H), 1.30 (t, J ) 7.3 Hz, 1.5H),
1.31 (t, J ) 7.3 Hz, 1.5H), 1.05-1.42 (m, 9H), 1.49-1.68 (m,
1H), 3.07-3.26 (m, 2H), 3.92-4.04 (m, 1H), 4.23 (q, J ) 7.3
Hz, 2H), 4.77-4.91 (m, 1H), 6.11 (d, J ) 8.3 Hz, 0.5H, NH),
6.20 (d, J ) 7.8 Hz, 0.5H, NH), 7.07-7.14 (m, 2H), 7.20-7.32
(m, 3H), 7.70 (d, J ) 8.3 Hz, 1H), 7.74 (d, J ) 8.3 Hz, 1H),
7.80 (d, J ) 9.8 Hz, 0.5H, NH), 7.88 (d, J ) 8.3 Hz, 1H), 7.94
(d, J ) 8.3 Hz, 1H), 7.86-7.96 (m, 0.5H, NH); 13C NMR (67.5
MHz, CDCl3) δ 14.0, 14.1, 22.5, 22.6, 22.96, 22.99, 25.1, 25.8,
26.4, 26.5, 29.1, 30.8, 31.0, 31.6, 37.4, 37.6, 45.1, 45.4, 52.66,
52.72, 57.56, 57.64, 61.8, 61.9, 114.67, 114.73, 118.2, 127.3,
127.4, 127.7, 128.6, 128.7, 129.0, 129.2, 132.3, 132.4, 135.4,
138.4, 138.6, 165.1, 165.2, 171.2, 171.8, 176.6, 177.1; MS (ESI)
m/z 506 (M + H)+, 528 (M + Na)+; HRMS (FAB) calcd for
N-[2,2-Dim eth yl-3-(N-(4-cya n oben zoyl)a m in o)-4-m eth -
ylp en ta n oyl]-L-p h en yla la n in e eth yl ester (8): 37% yield
1
from 5e; white foam; H NMR (270 MHz, CDCl3) δ 0.63 (d, J
) 6.8 Hz, 1.5H), 0.75 (d, J ) 6.8 Hz, 1.5H), 0.95 (br t, J ) 6.8
Hz, 3H), 1.225 (s, 1.5H), 1.233 (s, 1.5H), 1.26 (s, 1.5H), 1.271
(s, 1.5H), 1.273 (d, J ) 6.8 Hz, 1.5H), 1.31 (d, J ) 6.8 Hz, 1.5H),
1.93-2.15 (m, 1H), 3.07-3.26 (m, 2H), 3.95-4.02 (m, 1H),
4.13-4.28 (m, 2H), 4.78-4.88 (m, 1H), 6.19 (d, J ) 8.3 Hz,
0.5H, NH), 6.31 (d, J ) 7.3 Hz, 0.5H, NH), 7.07-7.18 (m, 2H),
7.22-7.36 (m, 3H), 7.73 (d, J ) 8.8 Hz, 1H), 7.76 (d, J ) 8.8
Hz, 1H), 7.97 (d, J ) 8.8 Hz, 1H), 8.00 (d, J ) 8.8 Hz, 1H),
8.49 (d, J ) 9.3 Hz, 0.5H, NH), 8.60 (d, J ) 9.3 Hz, 0.5H, NH);
13C NMR (67.5 MHz, CDCl3) δ 14.06, 14.09, 16.3, 16.4, 22.3,
22.4, 22.6, 22.8, 27.2, 27.7, 29.2, 29.3, 37.5, 37.7, 43.6, 43.9,
52.6, 52.8, 61.77, 61.84, 62.06, 62.13, 114.7, 118.2, 127.3, 127.4,
127.7, 128.6, 128.8, 129.0, 129.2, 132.37, 132.42, 135.4, 135.5,
138.5, 138.7, 165.47, 165.53, 171.2, 171.4, 177.7, 177.8; MS
(ESI) m/z 464 (M + H)+, 486 (M + Na)+. Anal. (C27H33N3O4)
C, H, N.
C
30H40N3O4 (M + H)+ 506.3019, found 506.2998. Anal.
N -[2,2-Dim e t h yl-3-(N -(4-cya n ob e n zoyl)a m in o)h e p -
ta n oyl]-L-p h en yla la n in e eth yl ester (9): 63% yield from 5f;
white foam; 1H NMR (270 MHz, CDCl3) δ 0.80-0.88 (m, 3H),
1.19 (s, 3H), 1.22 (s, 3H), 1.30 (t, J ) 7.3 Hz, 1.5H), 1.31 (t, J
) 7.3 Hz, 1.5H), 1.05-1.42 (m, 5H), 1.47-1.67 (m, 1H), 3.07-
3.27 (m, 2H), 3.92-4.04 (m, 1H), 4.23 (q, J ) 7.3 Hz, 2H), 4.77-
4.90 (m, 1H), 6.10 (d, J ) 8.3 Hz, 0.5H, NH), 6.19 (d, J ) 7.3
Hz, 0.5H, NH), 7.07-7.14 (m, 2H), 7.20-7.31 (m, 3H), 7.70
(d, J ) 8.8 Hz, 1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.81 (d, J ) 9.8
Hz, 0.5H, NH), 7.88 (d, J ) 8.8 Hz, 1H), 7.94 (d, J ) 8.8 Hz,
1H), 7.86-7.96 (m, 0.5H, NH); 13C NMR (67.5 MHz, CDCl3) δ
14.0, 14.1, 22.5, 23.0, 25.1, 25.8, 28.6, 28.7, 30.6, 30.7, 37.5,
37.6, 45.1, 45.4, 52.67, 52.74, 57.5, 57.6, 61.8, 61.9, 114.7, 114.8,
118.2, 127.3, 127.4, 127.7, 128.6, 128.7, 129.0, 129.2, 132.3,
132.4, 135.4, 138.4, 138.6, 165.1, 165.3, 171.3, 171.8, 176.6,
177.1; MS (ESI) m/z 478 (M + H)+, 500 (M + Na)+. Anal.
(C28H35N3O4‚0.25H2O) C, H, N.
(C30H39N3O4) C, H, N.
Compounds 2, 6-16, and 20-25 were prepared according
to general procedure C described for the preparation of 3
starting from the appropriate intermediates 5 by coupling with
L-phenylalanine methyl, ethyl, n-propyl, isopropyl, benzyl, and
tert-butyl esters.
N-[2,2-Dim et h yl-3-(N-(4-cya n ob en zoyl)a m in o)-5-p h e-
n ylp en ta n oyl]-L-p h en yla la n in e eth yl ester (2): 58% yield
from 5a ; colorless oil; 1H NMR (270 MHz, CDCl3) δ 1.20 (s,
3H), 1.22 (s, 3H), 1.24 (t, J ) 7.3 Hz, 1.5H), 1.30 (t, J ) 7.3
Hz, 1.5H), 1.38-1.65 (m, 1H), 1.77-1.98 (m, 1H), 2.54-2.72
(m, 2H), 3.04-3.24 (m, 2H), 4.01-4.13 (m, 1H), 4.14-4.28 (m,
2H), 4.75-4.88 (m, 1H), 6.06 (d, J ) 7.8 Hz, 0.5H, NH), 6.18
(d, J ) 7.8 Hz, 0.5H, NH), 7.03-7.31 (m, 10H), 7.71 (d, J )
8.8 Hz, 1H), 7.75 (d, J ) 8.8 Hz, 1H), 7.87 (d, J ) 8.8 Hz, 1H),
7.94 (d, J ) 8.8 Hz, 1H), 7.84-7.95 (m, 0.5H, NH), 8.04 (d, J
) 9.3 Hz, 0.5H, NH); MS (ESI) m/z 526 (M + H)+, 548 (M +
Na)+. Anal. (C32H35N3O4‚0.75H2O) C, H, N.
N-[2,2-Dim eth yl-3-(N-(4-cya n oben zoyl)a m in o)-5-m eth -
ylh exa n oyl]-L-p h en yla la n in e eth yl ester (10): 59% yield
1
from 5g; white foam; H NMR (270 MHz, CDCl3) δ 0.86 (d, J
N -[2,2-Dim e t h yl-3-(N -(4-cya n ob e n zoyl)a m in o)p e n -
ta n oyl]-L-p h en yla la n in e eth yl ester (6): 55% yield from 5c;
white foam; 1H NMR (270 MHz, CDCl3) δ 0.86 (t, J ) 7.3 Hz,
1.5H), 0.91 (t, J ) 7.3 Hz, 1.5H), 1.19 (s, 3H), 1.22 (s, 3H),
1.15-1.34 (m, 1H), 1.296 (t, J ) 7.3 Hz, 1.5H), 1.303 (t, J )
7.3 Hz, 1.5H), 1.54-1.78 (m, 1H), 3.07-3.25 (m, 2H), 3.86-
3.98 (m, 1H), 4.227 (q, J ) 7.3 Hz, 1H), 4.230 (q, J ) 7.3 Hz,
1H), 4.76-4.91 (m, 1H), 6.10 (d, J ) 8.3 Hz, 0.5H, NH), 6.19
(d, J ) 8.3 Hz, 0.5H, NH), 7.06-7.13 (m, 2H), 7.20-7.31 (m,
3H), 7.71 (d, J ) 8.3 Hz, 1H), 7.74 (d, J ) 8.3 Hz, 1H), 7.81 (d,
J ) 9.8 Hz, 0.5H, NH), 7.89 (d, J ) 8.3 Hz, 1H), 7.95 (d, J )
8.3 Hz, 1H), 7.86-7.98 (m, 0.5H, NH); 13C NMR (67.5 MHz,
CDCl3) δ 11.0, 11.1, 14.1, 22.9, 23.0, 23.8, 24.0, 25.2, 25.9, 37.5,
37.7, 45.0, 45.3, 52.6, 52.7, 59.0, 59.1, 61.8, 61.9, 114.7, 114.8,
118.2, 127.36, 127.39, 127.7, 128.66, 128.72, 129.1, 129.2,
132.3, 132.4, 135.4, 138.4, 138.6, 165.3, 165.5, 171.3, 171.8,
176.6, 177.1; MS (ESI) m/z 450 (M + H)+. Anal. (C26H31N3O4‚
H2O) C, H, N.
) 6.8 Hz, 3H), 0.93 (d, J ) 4.9 Hz, 1.5H), 0.95 (d, J ) 4.9 Hz,
1.5H), 1.19 (s, 3H), 1.22 (s, 3H), 1.15-1.37 (m, 2H), 1.30 (t, J
) 7.3 Hz, 1.5H), 1.31 (t, J ) 7.3 Hz, 1.5H), 1.49-1.67 (m, 1H),
3.08-3.25 (m, 2H), 4.02-4.13 (m, 1H), 4.23 (q, J ) 7.3 Hz,
2H), 4.77-4.91 (m, 1H), 6.08 (d, J ) 8.3 Hz, 0.5H, NH), 6.18
(d, J ) 7.3 Hz, 0.5H, NH), 7.07-7.13 (m, 2H), 7.20-7.32 (m,
3H), 7.70 (d, J ) 8.8 Hz, 1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.69-
7.83 (m, 1H, NH), 7.87 (d, J ) 8.8 Hz, 1H), 7.93 (d, J ) 8.8
Hz, 1H); 13C NMR (67.5 MHz, CDCl3) δ 14.1, 21.5, 23.0, 23.9,
24.0, 25.0, 25.1, 25.7, 37.5, 37.6, 40.1, 40.3, 45.3, 45.5, 52.66,
52.74, 55.8, 61.8, 62.0, 114.7, 114.8, 118.2, 127.3, 127.5, 127.7,
128.6, 128.7, 129.0, 129.2, 132.3, 132.4, 135.4, 135.5, 138.3,
138.6, 165.0, 165.2, 171.3, 171.9, 176.6, 177.1; MS (ESI) m/z
478 (M + H)+, 500 (M + Na)+. Anal. (C28H35N3O4‚H2O) C, H,
N.
N-[2,2-Dim eth yl-3-(N-(4-cyan oben zoyl)am in o)octan oyl]-
L-p h en yla la n in e eth yl ester (11): 88% yield from 5h ; white

A New Inhibitor of R-Chymotrypsin
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2 321
foam; 1H NMR (270 MHz, CDCl3) δ 0.83 (br t, J ) 6.8 Hz,
1.5H), 0.85 (br t, J ) 6.8 Hz, 1.5H), 1.19 (s, 3H), 1.22 (s, 3H),
1.30 (t, J ) 7.3 Hz, 1.5H), 1.31 (t, J ) 7.3 Hz, 1.5H), 1.08-
1.38 (m, 7H), 1.47-1.67 (m, 1H), 3.07-3.25 (m, 2H), 3.92-
4.04 (m, 1H), 4.23 (q, J ) 7.3 Hz, 2H), 4.78-4.90 (m, 1H), 6.10
(d, J ) 8.3 Hz, 0.5H, NH), 6.20 (d, J ) 7.8 Hz, 0.5H, NH),
7.08-7.14 (m, 2H), 7.20-7.34 (m, 3H), 7.70 (d, J ) 8.8 Hz,
1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.88 (d, J ) 8.8 Hz, 1H), 7.94 (d,
J ) 8.8 Hz, 1H), 7.78-7.97 (m, 1H, NH); 13C NMR (67.5 MHz,
CDCl3) δ 14.0, 14.1, 22.5, 22.97, 23.00, 25.1, 25.8, 26.1, 26.2,
30.8, 31.0, 31.6, 37.5, 37.6, 45.1, 45.4, 52.67, 52.74, 57.58, 57.64,
61.8, 61.9, 114.68, 114.74, 118.2, 127.3, 127.4, 127.7, 128.6,
128.7, 129.0, 129.2, 132.3, 132.4, 135.5, 138.4, 138.6, 165.1,
165.3, 171.3, 171.8, 176.6, 177.1; MS (ESI) m/z 492 (M + H)+,
514 (M + Na)+. Anal. (C29H37N3O4) C, H, N.
N-[2,2-Dim eth yl-3-(N-(4-cyan oben zoyl)am in o)decan oyl]-
L-p h en yla la n in e eth yl ester (12): 30% yield from 5i; white
solid; mp 107-109 °C; 1H NMR (270 MHz, CDCl3) δ 0.81-
0.88 (m, 3H), 1.22 (br s, 3H), 1.24 (br s, 3H), 1.30 (br t, J ) 6.8
Hz, 1.5H), 1.31 (br t, J ) 6.8 Hz, 1.5H), 1.16-1.39 (m, 11H),
1.48-1.67 (m, 1H), 3.08-3.27 (m, 2H), 3.97 (m, 1H), 4.23 (q,
J ) 6.8 Hz, 2H), 4.76-4.91 (m, 1H), 6.10 (d, J ) 7.8 Hz, 0.5H,
NH), 6.19 (d, J ) 7.8 Hz, 0.5H, NH), 7.07-7.30 (m, 5H), 7.68-
7.98 (m, 5H); 13C NMR (67.5 MHz, CDCl3) δ 14.0, 14.1, 22.56,
22.59, 22.97, 23.00, 25.1, 25.8, 26.5, 26.6, 29.1, 29.4, 30.9, 31.0,
31.7, 37.5, 37.6, 45.1, 45.4, 52.67, 52.74, 57.6, 57.7, 61.8, 61.9,
114.7, 118.2, 127.3, 127.4, 127.7, 128.6, 128.7, 129.0, 129.2,
132.3, 132.4, 135.5, 138.4, 138.6, 165.1, 165.3, 171.5, 171.8,
176.6, 177.1; MS (ESI) m/z 520 (M + H)+, 542 (M + Na)+. Anal.
(C31H41N3O4) C, H, N.
J ) 9.8 Hz, 0.5H, NH), 7.88 (d, J ) 8.8 Hz, 1H), 7.94 (d, J )
8.8 Hz, 1H), 7.85-7.95 (m, 0.5H, NH); 13C NMR (67.5 MHz,
CDCl3) δ 14.1, 22.7, 23.0, 25.1, 25.8, 26.5, 26.6, 29.3, 29.5, 29.6,
30.8, 31.0, 31.9, 37.5, 37.6, 45.1, 45.4, 52.67, 52.74, 57.6, 57.7,
61.8, 61.9, 114.7, 114.8, 118.2, 127.3, 127.4, 127.7, 128.6, 128.7,
129.0, 129.2, 132.3, 132.4, 135.4, 138.4, 138.6, 165.1, 165.3,
171.2, 171.8, 176.6, 177.1; MS (ESI) m/z 612 (M + Na)+. Anal.
(C36H51N3O4) C, H, N.
N-[2,2-Dim et h yl-3-(N-(4-b en zoyl)a m in o)n on a n oyl]-L-
p h en yla la n in e eth yl ester (16): 57% yield from 5m ; colorless
1
oil; H NMR (270 MHz, CDCl3) δ 0.79-0.88 (m, 3H), 1.21 (s,
3H), 1.22 (s, 3H), 1.29 (t, J ) 7.3 Hz, 3H), 1.10-1.40 (m, 9H),
1.45-1.67 (m, 1H), 3.07-3.25 (m, 2H), 3.96-4.09 (m, 1H), 4.22
(q, J ) 7.3 Hz, 2H), 4.79-4.91 (m, 1H), 6.12 (d, J ) 7.8 Hz,
0.5H, NH), 6.19 (d, J ) 7.8 Hz, 0.5H, NH), 7.07-7.15 (m, 2H),
7.20-7.32 (m, 3H), 7.37-7.51 (m, 3H), 7.59 (d, J ) 9.8 Hz,
0.5H, NH), 7.65 (d, J ) 9.8 Hz, 0.5H, NH), 7.78-7.87 (m, 2H);
MS (ESI) m/z 481 (M + H)+, 503 (M + Na)+. Anal. (C29H40N2O4‚
0.5H2O) C, H, N.
N -[2,2-D im e t h y l-3-(N -(b u t y lo x y c a r b o n y l)a m in o )-
n on a n oyl]-L-p h en yla la n in e eth yl ester (21): 98% yield
from 5n ; white solid; mp 82-84 °C; 1H NMR (270 MHz, CDCl3)
δ 0.86 (t, J ) 6.8 Hz, 1.5H), 0.87 (t, J ) 6.8 Hz, 1.5H), 1.14 (br
s, 3H), 1.15 (br s, 3H), 0.92-1.48 (m, 10H), 1.26 (t, J ) 6.8
Hz, 3H), 1.41 (s, 4.5H), 1.44 (s, 4.5H), 3.00-3.23 (m, 2H), 3.36-
3.48 (m, 1H), 4.19 (q, J ) 6.8 Hz, 2H), 4.76-4.87 (m, 1H), 5.11
(d, J ) 10.3 Hz, 0.5H, NH), 5.33 (d, J ) 10.3 Hz, 0.5H, NH),
6.00-6.13 (m, 1H, NH), 7.09-7.15 (m, 2H), 7.21-7.34 (m, 3H);
13C NMR (67.5 MHz, CDCl3) δ 14.1, 22.5, 22.6, 23.3, 23.8, 23.9,
26.5, 26.6, 28.36, 28.40, 29.0, 29.1, 31.1, 31.8, 37.7, 37.8, 45.8,
45.9, 52.8, 57.9, 58.1, 61.5, 61.6, 78.6, 78.7, 127.16, 127.19,
128.6, 129.1, 129.2, 135.8, 135.9, 156.4, 171.6, 171.7, 176.2,
176.3; MS (ESI) m/z 477 (M + H)+, 499 (M + Na)+. Anal.
(C27H44N2O5) C, H, N.
N-[2,2-Dim et h yl-3-(N-(4-cya n ob en zoyl)a m in o)u n d e-
ca n oyl]-L-p h en yla la n in e eth yl ester (13): 49% yield from
5j; white solid; mp 114 °C; 1H NMR (270 MHz, CDCl3) δ 0.84
(br t, J ) 6.8 Hz, 1.5H), 0.85 (br t, J ) 6.8 Hz, 1.5H), 1.19 (s,
3H), 1.21 (s, 3H), 1.30 (t, J ) 7.3 Hz, 1.5H), 1.31 (t, J ) 7.3
Hz, 1.5H), 1.04-1.43 (m, 13H), 1.47-1.68 (m, 1H), 3.07-3.24
(m, 2H), 3.91-4.05 (m, 1H), 4.23 (q, J ) 7.3 Hz, 2H), 4.77-
4.90 (m, 1H), 6.10 (d, J ) 7.3 Hz, 0.5H, NH), 6.19 (d, J ) 7.3
Hz, 0.5H, NH), 7.07-7.14 (m, 2H), 7.20-7.34 (m, 3H), 7.70
(d, J ) 8.3 Hz, 1H), 7.77 (d, J ) 8.3 Hz, 1H), 7.88 (d, J ) 8.3
N -[2 ,2 -D i m e t h y l -3 -(N -(4 -c y a n o b e n z o y l )a m i n o )-
n on a n oyl]-L-p h en yla la n in e m eth yl ester (22): 61% yield
1
from 5b; colorless oil; H NMR (270 MHz, CDCl3) δ 0.83 (t, J
) 6.8 Hz, 1.5H), 0.85 (t, J ) 6.8 Hz, 1.5H), 1.19 (s, 3H), 1.22
(s, 3H), 1.05-1.34 (m, 9H), 1.45-1.65 (m, 1H), 3.05-3.26 (m,
2H), 3.79 (s, 3H), 3.91-4.06 (m, 1H), 4.80-4.92 (m, 1H), 6.10
(d, J ) 8.4 Hz, 0.5H, NH), 6.18 (d, J ) 7.3 Hz, 0.5H, NH),
7.06-7.13 (m, 2H), 7.20-7.32 (m, 3H), 7.71 (d, J ) 8.8 Hz,
1H), 7.74 (d, J ) 8.3 Hz, 1H), 7.88 (d, J ) 8.8 Hz, 1H), 7.94 (d,
J ) 8.8 Hz, 1H), 7.73-7.95 (m, 1H, NH); 13C NMR (67.5 MHz,
CDCl3) δ 14.0, 22.5, 22.6, 23.0, 25.1, 25.8, 26.4, 26.5, 29.2, 30.9,
31.0, 31.6, 37.4, 37.6, 45.1, 45.4, 52.55, 52.66, 52.71, 57.58,
57.64, 114.7, 114.8, 118.2, 127.4, 127.5, 127.9, 128.7, 128.8,
129.0, 129.1, 132.3, 132.4, 135.4, 138.4, 138.6, 165.1, 165.3,
171.7, 172.2, 176.7, 177.2; MS (ESI) m/z 492 (M + H)+, 514
(M + Na)+. Anal. (C29H37N3O4‚0.5H2O) C, H, N.
Hz, 1H), 7.94 (d, J ) 8.3 Hz, 1H), 7.75-7.98 (m, 1H, NH); 13
C
NMR (67.5 MHz, CDCl3) δ 14.08, 14.12, 22.6, 23.0, 25.1, 25.9,
26.5, 26.6, 29.2, 29.4, 29.5, 30.9, 31.0, 31.8, 37.5, 37.6, 45.1,
45.4, 52.67, 52.74, 57.6, 57.7, 61.8, 61.9, 114.68, 114.74, 118.2,
127.3, 127.4, 127.7, 128.6, 128.7, 129.0, 129.2, 132.3, 132.4,
135.5, 138.4, 138.6, 165.1, 165.3, 171.3, 171.8, 176.6, 177.1;
MS (ESI) m/z 534 (M + H)+, 556 (M + Na)+. Anal. (C32H43N3O4)
C, H, N.
N-[2,2-Dim et h yl-3-(N-(4-cya n ob en zoyl)a m in o)t r id e-
ca n oyl]-L-p h en yla la n in e eth yl ester (14): 87% yield from
1
5k ; waxy solid; H NMR (270 MHz, CDCl3) δ 0.86 (br t, J )
N -[2 ,2 -D i m e t h y l -3 -(N -(4 -c y a n o b e n z o y l )a m i n o )-
n on a n oyl]-L-p h en yla la n in e n -p r op yl ester (23): 99% yield
6.8 Hz, 3H), 1.19 (s, 3H), 1.12-1.37 (m, 20H), 1.30 (t, J ) 7.3
Hz, 1.5H), 1.31 (t, J ) 7.3 Hz, 1.5H), 1.47-1.67 (m, 1H), 3.07-
3.25 (m, 2H), 3.90-4.03 (m, 1H), 4.23 (q, J ) 7.3 Hz, 2H), 4.77-
4.90 (m, 1H), 6.10 (d, J ) 7.3 Hz, 0.5H, NH), 6.19 (d, J ) 8.3
Hz, 0.5H, NH), 7.07-7.13 (m, 2H), 7.19-7.33 (m, 3H), 7.70
(d, J ) 8.8 Hz, 1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.88 (d, J ) 8.8
1
from 5b; colorless oil; H NMR (270 MHz, CDCl3) δ 0.83 (t, J
) 6.8 Hz, 1.5H), 0.85 (t, J ) 6.8 Hz, 1.5H), 0.95 (t, J ) 7.3 Hz,
1.5H), 0.96 (t, J ) 7.3 Hz, 1.5H), 1.19 (s, 3H), 1.21 (s, 3H),
1.04-1.47 (m, 9H), 1.48-1.68 (m, 1H), 1.62-1.75 (m, 2H),
3.07-3.26 (m, 2H), 3.92-4.03 (m, 1H), 4.13 (t, J ) 6.8 Hz, 2H),
4.79-4.92 (m, 1H), 6.11 (d, J ) 7.3 Hz, 0.5H, NH), 6.20 (d, J
) 7.8 Hz, 0.5H, NH), 7.07-7.14 (m, 2H), 7.19-7.33 (m, 3H),
7.70 (d, J ) 8.8 Hz, 1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.88 (d, J )
8.8 Hz, 1H), 7.94 (d, J ) 8.8 Hz, 1H), 7.81 (br d, J ) 9.8 Hz,
0.5H, NH), 7.87-7.96 (m, 0.5H, NH); 13C NMR (67.5 MHz,
CDCl3) δ 10.3, 14.0, 21.8, 22.5, 22.6, 22.96, 22.99, 25.1, 25.8,
26.4, 26.5, 29.1, 30.8, 31.0, 31.6, 37.5, 37.7, 45.1, 45.4, 52.67,
52.72, 57.58, 57.64, 67.3, 67.5, 114.67, 114.73, 118.2, 127.3,
127.4, 127.7, 128.6, 128.7, 129.0, 129.2, 132.3, 132.4, 135.4,
138.4, 138.6, 165.1, 165.2, 171.3, 171.9, 176.6, 177.1; MS (ESI)
m/z 520 (M + H)+, 542 (M + Na)+. Anal. (C31H41N3O4‚2H2O)
C, H, N.
Hz, 1H), 7.94 (d, J ) 8.8 Hz, 1H), 7.77-7.97 (m, 1H, NH); 13
C
NMR (67.5 MHz, CDCl3) δ 14.09, 14.12, 22.7, 22.97, 23.00,
25.1, 25.8, 26.5, 26.6, 29.3, 29.5, 29.6, 30.9, 31.0, 31.9, 37.5,
37.6, 45.1, 45.4, 52.67, 52.74, 57.6, 57.7, 61.8, 61.9, 114.68,
114.74, 118.2, 127.3, 127.4, 127.7, 128.6, 128.7, 129.0, 129.2,
132.3, 132.4, 135.5, 138.4, 138.6, 165.1, 165.3, 171.3, 171.8,
176.6, 177.1; MS (ESI) m/z 562 (M + H)+, 584 (M + Na)+. Anal.
(C34H47N3O4‚H2O) C, H, N.
N-[2,2-Dim eth yl-3-(N-(4-cya n oben zoyl)a m in o)p en ta d e-
ca n oyl]-L-p h en yla la n in e eth yl ester (15): 71% yield from
1
5l; waxy solid; H NMR (270 MHz, CDCl3) δ 0.87 (br t, J )
6.8 Hz, 3H), 1.19 (br s, 3H), 1.05-1.43 (m, 24H), 1.30 (t, J )
7.3 Hz, 1.5H), 1.31 (t, J ) 7.3 Hz, 1.5H), 1.47-1.68 (m, 1H),
3.07-3.26 (m, 2H), 3.91-4.04 (m, 1H), 4.23 (q, J ) 7.3 Hz,
2H), 4.77-4.90 (m, 1H), 6.10 (d, J ) 8.3 Hz, 0.5H, NH), 6.19
(d, J ) 7.3 Hz, 0.5H, NH), 7.07-7.13 (m, 2H), 7.19-7.31 (m,
3H), 7.70 (d, J ) 8.8 Hz, 1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.81 (d,
N -[2 ,2 -D i m e t h y l -3 -(N -(4 -c y a n o b e n z o y l )a m i n o )
n on a n oyl]-L-p h en yla la n in e isop r op yl ester (24): 84% yield
1
from 5b; colorless oil; H NMR (270 MHz, CDCl3) δ 0.83 (t, J
) 6.8 Hz, 1.5H), 0.85 (t, J ) 6.8 Hz, 1.5H), 1.19 (s, 3H), 1.22
(s, 3H), 1.07-1.48 (m, 15H), 1.50-1.70 (m, 1H), 3.07-3.24 (m,

322 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 2
Iijima et al.
2H), 3.91-4.05 (m, 1H), 4.74-4.87 (m, 1H), 5.00-5.12 (m, 1H),
6.10 (d, J ) 7.8 Hz, 0.5H, NH), 6.20 (d, J ) 7.3 Hz, 0.5H, NH),
7.08-7.15 (m, 2H), 7.19-7.31 (m, 3H), 7.70 (d, J ) 8.8 Hz,
1H), 7.74 (d, J ) 8.8 Hz, 1H), 7.83 (d, J ) 9.8 Hz, 0.5H, NH),
7.88 (d, J ) 8.8 Hz, 1H), 7.94 (d, J ) 8.8 Hz, 1H), 7.88-7.97
(m, 0.5H, NH); MS (ESI) m/z 520 (M + H)+, 542 (M + Na)+.
Anal. (C31H41N3O4‚H2O) C, H, N.
n o)n on a n oyl]-L-p h en yla la n in e eth yl ester (19): 60% yield
from 21; colorless oil; 1H NMR (270 MHz, CDCl3) δ 0.86 (br t,
J ) 6.8 Hz, 3H), 1.19-1.40 (m, 15H), 1.27 (br t, J ) 7.3 Hz,
3H), 1.42-1.60 (m, 1H), 3.04-3.22 (m, 2H), 3.94-4.06 (m, 1H),
4.19 (q, J ) 7.3 Hz, 2H), 4.80-4.91 (m, 1H), 6.11 (d, J ) 8.3
Hz, 0.5H, NH), 6.20 (d, J ) 7.8 Hz, 0.5H, NH), 7.07-7.15 (m,
2H), 7.22-7.32 (m, 3H), 7.37-7.45 (m, 1H), 7.50-7.59 (m, 1H),
7.95-8.20 (m, 2H); MS (ESI) m/z 524 (M + H)+, 546 (M + Na)+.
Anal. (C30H38FN3O4‚0.5H2O) C, H, N.
N -[2,2-Dim e t h yl-3-(N -(2-fu r oyl)a m in o)n on a n oyl]-L-
p h en yla la n in e eth yl ester (20): 51% yield from 21; pale-
yellow oil; 1H NMR (270 MHz, CDCl3) δ 0.83 (br t, J ) 6.8 Hz,
1.5H), 0.85 (br t, J ) 6.8 Hz, 1.5H), 1.19 (br s, 3H), 1.22 (br s,
3H), 1.28 (br t, J ) 7.3 Hz, 3H), 1.06-1.38 (m, 9H), 1.44-1.65
(m, 1H), 3.04-3.25 (m, 2H), 3.90-4.03 (m, 1H), 4.21 (q, J )
7.3 Hz, 2H), 4.80-4.92 (m, 1H), 6.14 (d, J ) 7.8 Hz, 0.5H, NH),
6.20 (d, J ) 7.8 Hz, 0.5H, NH), 6.46-6.56 (m, 1H), 7.07-7.33
(m, 6H), 7.44-7.65 (m, 2H); MS (ESI) m/z 472 (M + H)+, 493
(M + Na)+. Anal. (C27H38N2O5‚0.25H2O) C, H, N.
N -[2 ,2 -D i m e t h y l -3 -(N -(4 -c y a n o b e n z o y l )a m i n o )-
n on a n oyl]-L-p h en yla la n in e ben zyl ester (25): 92% yield
1
from 5b; colorless oil; H NMR (270 MHz, CDCl3) δ 0.84 (t, J
) 6.8 Hz, 1.5H), 0.85 (t, J ) 6.8 Hz, 1.5H), 1.18 (s, 3H), 1.19
(s, 3H), 1.16-1.40 (m, 9H), 1.49-1.68 (m, 1H), 3.06-3.24 (m,
2H), 3.91-4.02 (m, 1H), 4.82-4.96 (m, 1H), 5.10-5.30 (m, 2H),
6.08 (d, J ) 7.8 Hz, 0.5H, NH), 6.18 (d, J ) 7.8 Hz, 0.5H, NH),
6.93-7.00 (m, 2H), 7.15-7.26 (m, 2H), 7.32-7.42 (m, 6H), 7.68
(d, J ) 8.3 Hz, 1H), 7.73 (d, J ) 8.8 Hz, 1H), 7.73-7.79 (m,
0.5H, NH), 7.85 (d, J ) 8.3 Hz, 1H), 7.93 (d, J ) 8.8 Hz, 1H),
7.84-7.92 (m, 0.5H, NH); MS (ESI) m/z 566 (M - H)+. Anal.
(C35H41N3O4‚2H2O) C, H, N.
N -[2 ,2 -D i m e t h y l -3 -(N -(4 -c y a n o b e n z o y l )a m i n o )-
n on a n oyl]-L-p h en yla la n in e ter t-bu tyl ester (26): 55% yield
from 5b; white solid; mp 174-176 °C; 1H NMR (270 MHz,
CDCl3) δ 0.83 (br t, J ) 6.8 Hz, 3H), 1.19 (s, 3H), 1.22 (s, 3H),
1.15-1.36 (m, 9H), 1.46 (s, 4.5H), 1.47 (s, 4.5H), 1.54-1.68
(m, 1H), 3.04-3.21 (m, 2H), 3.92-4.04 (m, 1H), 4.66-4.81 (m,
1H), 6.10 (d, J ) 7.8 Hz, 0.5H, NH), 6.22 (d, J ) 7.8 Hz, 0.5H,
NH), 7.10-7.33 (m, 5H), 7.69 (d, J ) 8.3 Hz, 1H), 7.74 (d, J )
8.3 Hz, 1H), 7.89 (d, J ) 8.3 Hz, 1H), 7.94 (d, J ) 8.3 Hz, 1H),
7.80-8.00 (m, 1H, NH); MS (ESI) m/z 535 (M + H)+, 556 (M
+ Na)+. Anal. (C32H43N3O4) C, H, N.
Ack n ow led gm en t. The authors thank Dr. Yoichi
Matsuzaki for helpful discussions and suggestions and
also thank Kiyoshi Morita for his support in analytical
chemistry.
Su p p or tin g In for m a tion Ava ila ble: Figures 3 and 4 and
1H and 13C NMR spectral data for all synthetic intermediates.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Gen er a l P r oced u r e D for P r ep a r a tion of Ta r get Com -
p ou n d s. N-[2,2-Dim eth yl-3-(N-(3,4-d iflu or oben zoyl)a m i-
n o)n on a n oyl]-L-p h en yla la n in e Eth yl Ester (18). To 21
(0.11 g, 0.23 mmol) was added 4 N HCl-dioxane (10 mL). The
reaction mixture was stirred for 1 h under cooling with ice.
After removal of the solvent in vacuo at room temperature,
the resulting residue was washed three times with hexane and
dissolved in CH2Cl2 (10 mL) under cooling with ice. After the
mixture was neutralized by triethylamine, 3,4-difluorobenzoic
acid (40 mg, 0.23 mmol), HOBt (40 mg, 0.26 mmol), and
WSCD‚HCl (60 mg, 0.3 mmol) were added, and the mixture
was stirred overnight. After removal of the solvent by evapora-
tion, the resulting residue was dissolved in EtOAc (30 mL),
washed with 5% citric acid (3 × 30 mL), 5% NaHCO3 (3 × 30
mL), and saturated NaCl (3 × 30 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by column
chromatography on silica (1.8 × 20 cm) using hexanes-EtOAc
(6:1 to 1:1) as an eluent to give an oil of 18 (36 mg, 30%):
colorless oil; 1H NMR (270 MHz, CDCl3) δ 0.83 (t, J ) 6.8 Hz,
1.5H), 0.85 (t, J ) 6.8 Hz, 1.5H), 1.18 (s, 3H), 1.20 (s, 3H),
1.30 (t, J ) 7.3 Hz, 1.5H), 1.31 (t, J ) 7.3 Hz, 1.5H), 1.04-
1.38 (m, 9H), 1.47-1.66 (m, 1H), 3.07-3.25 (m, 2H), 3.89-
4.01 (m, 1H), 4.18-4.29 (m, 2H), 4.76-4.91 (m, 1H), 6.06 (d,
J ) 8.3 Hz, 0.5H, NH), 6.17 (d, J ) 8.3 Hz, 0.5H, NH), 7.07-
7.34 (m, 5H), 7.50-7.75 (m, 3H); MS (ESI) m/ z 517 (M + H)+.
Anal. (C29H38F2N2O4‚0.5H2O) C, H, N.
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1
from 21; colorless oil; H NMR (270 MHz, CDCl3) δ 0.83 (t, J
) 6.8 Hz, 1.5H), 0.85 (t, J ) 6.8 Hz, 1.5H), 1.17 (s, 3H), 1.19
(s, 3H), 1.30 (t, J ) 7.3 Hz, 1.5H), 1.32 (t, J ) 7.3 Hz, 1.5H),
1.05-1.38 (m, 9H), 1.50-1.67 (m, 1H), 3.07-3.25 (m, 2H),
3.90-4.02 (m, 1H), 4.18-4.31 (m, 2H), 4.78-4.91 (m, 1H), 6.03
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7.90 (d, J ) 2.4 Hz, 0.5H), 7.95 (d, J ) 2.4 Hz, 0.5H); MS (ESI)
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N-[2,2-Dim et h yl-3-(N-(2-flu or o-4-cya n ob en zoyl)a m i-

A New Inhibitor of R-Chymotrypsin
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